The Impact of ExoS on Pseudomonas aeruginosa Internalization by Epithelial Cells Is Independent of fleQ and Correlates with Bistability of Type Three Secretion System Gene Expression.
The Impact of ExoS on Pseudomonas aeruginosa Internalization by Epithelial Cells Is Independent of fleQ and Correlates with Bistability of Type Three Secretion System Gene Expression.
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DOI：
10.1128/mbio.00668-18
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发表时间：
2018-05-01
期刊：
影响因子：
6.4
通讯作者：
Fleiszig SMJ
中科院分区：
文献类型：
--
作者：
Kroken AR;Chen CK;Evans DJ;Yahr TL;Fleiszig SMJ
铜绿假单胞菌在体外和体内被内化到多种类型的上皮细胞中，但通常被认为是一种排他的细胞外病原体。特异性地，ExoS，一种第三型分泌系统（T3 SS）效应物，具有抗吞噬活性，但对于铜绿假单胞菌的细胞内存活及其在上皮细胞中的泡小生境的占据是必需的。在这里，我们使用侵入性（表达ExoS的）铜绿假单胞菌和相应的无效效应子同基因T3 SS突变体、具有和不具有ExoS转化的细胞毒性铜绿假单胞菌的无效效应子突变体、抗生素排除测定和使用T3 SS-GFP报告子的成像来解决这种二分法的机制。除了无效效应子PA 103之外，所有菌株在编码ExoS时都被内化。胞内细菌表现出T3 SS激活，在复制的子细胞中继续。校正无效效应子PA 103中的fleQ突变在有或没有ExoS的情况下促进内化>10倍。相反，在PAO 1中突变fleQ使内化减少>10倍，也有或没有ExoS。与与fleQ状态匹配的PAO 1相比，无表达载体的PA 103保持较不好的内化，但仅具有ExoS表达，这表明这些菌株之间存在其他差异。使用GFP荧光和定量逆转录-PCR（qRT-PCR）定量T3 SS活化显示，与其他分离株相比，菌株PA 103 ΔexoUT的T3 SS表达是高度可诱导的，并且与fleQ状态无关。这些发现支持了铜绿假单胞菌不完全是细胞外病原体的原理，在宿主细胞相互作用期间，内化受到群体中T3 SS阳性和T3 SS阴性细菌的相对比例的影响。这些数据也挑战了目前关于T3 SS效应子递送到宿主细胞中的想法，并表明T3 SS双稳态是研究铜绿假单胞菌发病机制的重要考虑因素。铜绿假单胞菌通常被称为细胞外病原体，尽管其被证明具有侵入宿主细胞并在宿主细胞内存活的能力。铜绿假单胞菌编码具有抗内化活性的T3 SS效应子，矛盾的是，这些效应子在细胞内存活中起关键作用。在这里，我们试图解决为什么ExoS不能阻止天然编码它的铜绿假单胞菌菌株的内化。结果表明，ExoS在通常用于研究T3 SS效应子活性的菌株PA 103的效应子无效背景下表达的条件下，表现出异常强的抗内化活性。抑制内化与T3 SS高诱导和ExoS递送相关。PA 103 fleQ突变，阻止鞭毛组装，进一步减少内化，但这样做独立于ExoS。结果显示所有菌株的细胞内T3 SS表达，并表明T3 SS双稳态影响铜绿假单胞菌内化。这些发现调和了文献中围绕铜绿假单胞菌内化的争议，并支持铜绿假单胞菌不完全是细胞外病原体的原则。
Pseudomonas aeruginosa is internalized into multiple types of epithelial cell in vitro and in vivo and yet is often regarded as an exclusively extracellular pathogen. Paradoxically, ExoS, a type three secretion system (T3SS) effector, has antiphagocytic activities but is required for intracellular survival of P. aeruginosa and its occupation of bleb niches in epithelial cells. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing) P. aeruginosa and corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxic P. aeruginosa with and without ExoS transformation, antibiotic exclusion assays, and imaging using a T3SS-GFP reporter. Except for effector-null PA103, all strains were internalized while encoding ExoS. Intracellular bacteria showed T3SS activation that continued in replicating daughter cells. Correcting the fleQ mutation in effector-null PA103 promoted internalization by >10-fold with or without ExoS. Conversely, mutating fleQ in PAO1 reduced internalization by >10-fold, also with or without ExoS. Effector-null PA103 remained less well internalized than PAO1 matched for fleQ status, but only with ExoS expression, suggesting additional differences between these strains. Quantifying T3SS activation using GFP fluorescence and quantitative reverse transcription-PCR (qRT-PCR) showed that T3SS expression was hyperinducible for strain PA103ΔexoUT versus other isolates and was unrelated to fleQ status. These findings support the principle that P. aeruginosa is not exclusively an extracellular pathogen, with internalization influenced by the relative proportions of T3SS-positive and T3SS-negative bacteria in the population during host cell interaction. These data also challenge current thinking about T3SS effector delivery into host cells and suggest that T3SS bistability is an important consideration in studying P. aeruginosa pathogenesis. P. aeruginosa is often referred to as an extracellular pathogen, despite its demonstrated capacity to invade and survive within host cells. Fueling the confusion, P. aeruginosa encodes T3SS effectors with anti-internalization activity that, paradoxically, play critical roles in intracellular survival. Here, we sought to address why ExoS does not prevent internalization of the P. aeruginosa strains that natively encode it. Results showed that ExoS exerted unusually strong anti-internalization activity under conditions of expression in the effector-null background of strain PA103, often used to study T3SS effector activity. Inhibition of internalization was associated with T3SS hyperinducibility and ExoS delivery. PA103 fleQ mutation, preventing flagellar assembly, further reduced internalization but did so independently of ExoS. The results revealed intracellular T3SS expression by all strains and suggested that T3SS bistability influences P. aeruginosa internalization. These findings reconcile controversies in the literature surrounding P. aeruginosa internalization and support the principle that P. aeruginosa is not exclusively an extracellular pathogen.